Figure 2

Establishment of in vivo mouse model of GR50 expression under a ubiquitous promoter system. (A) Schematic of the FAST cassette used to encode ATG-driven FLAG-GR50-GFP or FLAG-GFP in mice at the Rosa26 locus under the ROSA26 promoter. (B) Representative western blot probing for GR (top) or GFP (bottom) in either GR50-GFP or control-GFP mice. Lysate from human embryonic kidney (HEK) cells transiently transfected with FLAG-GR50-GFP used as a positive control. (C) Representative image of endogenous expression of GR50-GFP in the lumbar spinal cord and cortex of a 3-month-old GR50-GFP mouse. Enlargement scale bars = 50 µm. Selected cortex area approximates region used for subsequent quantifications. (D) Representative images of S100β-, Iba1-, or NeuN-positive CNS cells expressing GR50-GFP in 3-month-old mice. Percentage of astrocytes, microglia, and neurons positive for GR50-GFP within cortical layers (E) and lumbar spinal cord (F). One-way ANOVA, Tukey’s multiple comparison’s test. (E) Cortex: n = 3 mice; mean ± s.e.m. Astrocytes vs. microglia p = 0.9972, neurons vs. astrocytes p < 0.0001, neurons vs. microglia p < 0.0001. (F) Lumbar Spinal Cord: n = 3 mice; mean ± s.e.m. Astrocytes vs. microglia p = 0.2246, neurons vs. astrocytes p = 0.0013, neurons vs. microglia p = 0.0071. (G) Representative images of the localization patterns of GR50 within CNS at 3-months-old. Type I: diffuse, cytosolic. Type II: cytosolic with evidence of perinuclear accumulation. Type III: cytosolic, aggregated. Percent distribution of localization types within the cortex and lumbar spinal cord quantified in (H), (I), n = 222 cells from cortex, 164 cells from lumbar spinal cord from 4 mice (2m, 2f). Localization types were scored manually with experimenter blinded to the genotype of the mice.